Infections with Mollicutes species (such as Mycoplasma, Acholeplasma, and Ureaplasma) can induce a variety of problems in living organisms and laboratory cell cultures. Therefore, it is necessary to establish a routine diagnostic protocol for Mycoplasma infection in order to ensure reliable research results, as well as the safety of commercial biological products. For that purpose a novel PCR-based procedure using specific designed primers complementary to 16S rRNA genome region of mollicute species was evaluated. PCR was optimized and sensitivity and specificity was evaluated by defined cell count concentrations (2-31250 CFU/ml) of different strains of Mycoplasma, Acholeplasma and Ureaplasma. Amplicon (272 bp) was cloned by PCR-cloning and sequenced by dideoxy chain termination. PCR, was found to be able to detect 10 copies of mollicute target DNA. No cross-reactivity with genomic DNA of non-mollicute bacteria or human cell lines was observed. Forty seven human and animal cell lines were evaluated for mollicute contamination. Twenty five cell lines (53%) were correctly identified as contaminated by this molecular approach. The results of this study demonstrated that this PCR-based method is not only fast and reproducible, but also highly sensitive and specific for detecting contaminant mycoplasmas in cell cultures.
Background: In Congenital Disorder of Glycosylation (CDG) type Ia, homozygous mutations of the PMM2 gene cause phosphomannomutase 2 dysfunction. Case presentation: Herein, a 10-month-old girl is presented with severe hypotonia along with inappropriately normal mental status and normal facies. High 2-ketoglutaric acid was detected in her urine; therefore the diagnosis of 2-Ketoglutarate dehydrogenase complex (KDHC) deficiency was made for this patient. High dose of vitamin B1 was administered, because thiamine is considered as a co-factor in this inborn error of metabolism. She responded very well to daily administration of 500 mg/day vitamin B1 and stood up without help 5 months later. She had experienced seizure, which responded well to pyridoxine. Now, she is a 3.5-years-old child, who could talk and walk normally. Recently, whole exome sequencing was performed for her, which showed homozygote mutation of PMM2; therefore the diagnosis was changed from KDHC deficiency to PMM2-CDG. Conclusion: Attention to the pathophysiology of inborn errors of metabolism is necessary, while considering the defective enzymes co-factor may help us to find an option for treatment of such rare diseases.
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